Uses of novel fatty acid desaturases and elongases and products thereof

ABSTRACT

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturases and elongases from the organism Emiliana huxleyi. The invention also provides recombinant expression vectors containing desaturase or elongase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g. arachidonic acid (ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/351,962 filed Nov. 16, 2016, now U.S. Pat. No. 10,351,870, which is acontinuation of U.S. application Ser. No. 13/384,277 filed Jan. 16,2012, now U.S. Pat. No. 9,493,520, which is a national stage application(under 35 U.S.C. § 371) of PCT/EP2010/060178, filed Jul. 15, 2010, whichclaims benefit of U.S. Provisional Application No. 61/226,301, filedJul. 17, 2009, and European Application No. 09165752.8, filed Jul. 17,2009. The entire contents of each of these applications are herebyincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is 074021_0167_02_590745_5 T25. The size of thetext file is 59,186 bytes and the text file was created on Jun. 24,2019.

The invention in principle pertains to the field of recombinantmanufacture of fatty acids. It provides nucleic acid molecules whichencode novel fatty acid desaturases and elongases. The invention alsoprovides recombinant expression vectors containing desaturase andelongase nucleic acid molecules, host cells into which the expressionvectors have been introduced, and methods for large-scale production oflong chain polyunsaturated fatty acids (LCPUFAs), e.g. arachidonic acid(ARA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).

Fatty acids are carboxylic acids with long-chain hydrocarbon side groupsthat play a fundamental role in many biological processes. Fatty acidsare rarely found free in nature but, rather, occur in esterified form asthe major component of lipids. As such, lipids/fatty acids are sourcesof energy (e.g., b-oxidation). In addition, lipids/fatty acids are anintegral part of cell membranes and, therefore, are indispensable forprocessing biological or biochemical information.

Fatty acids can be divided into two groups: saturated fatty acids formedof single carbon bonds and the unsaturated fatty acids which contain oneor more carbon double bonds in cis-configuration. Unsaturated fattyacids are produced by terminal desaturases that belong to the class ofnonheme-iron enzymes. Each of these enzymes are part of anelectron-transport system that contains two other proteins, namelycytochrome b₅ and NADH-cytochrome b₅ reductase. Specifically, suchenzymes catalyze the formation of double bonds between the carbon atomsof a fatty acid molecule, for example, by catalyzing theoxygen-dependent dehydrogenation of fatty acids (Sperling et al., 2003).Human and other mammals have a limited spectrum of desaturases that arerequired for the formation of particular double bonds in unsaturatedfatty acids and thus, have a limited capacity for synthesizing essentialfatty acids, e.g., long chain polyunsaturated fatty acids (LCPUFAs).Thus, humans have to take up some fatty acids through their diet. Suchessential fatty acids include, for example, linoleic acid (C18:2),linolenic acid (C18:3). In contrast, insects, microorganisms and plantsare able to synthesize a much larger variety of unsaturated fatty acidsand their derivatives. Indeed, the biosynthesis of fatty acids is amajor activity of plants and microorganisms.

Long chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoicacid (DHA, 22:6(4,7,10,13,16,19)) are essential components of cellmembranes of various tissues and organelles in mammals (nerve, retina,brain and immune cells). For example, over 30% of fatty acids in brainphospholipid are 22:6 (n-3) and 20:4 (n-6) (Crawford, M. A., et al.,(1997) Am. J. Clin. Nutr. 66:1032S-1041S). In retina, DHA accounts formore than 60% of the total fatty acids in the rod outer segment, thephotosensitive part of the photoreceptor cell (Giusto, N. M., et al.(2000) Prog. Lipid Res. 39:315-391). Clinical studies have shown thatDHA is essential for the growth and development of the brain in infants,and for maintenance of normal brain function in adults (Martinetz, M.(1992) J. Pediatr. 120:S129-S138). DHA also has significant effects onphotoreceptor function involved in the signal transduction process,rhodopsin activation, and rod and cone development (Giusto, N. M., etal. (2000) Prog. Lipid Res. 39:315-391). In addition, some positiveeffects of DHA were also found on diseases such as hypertension,arthritis, atherosclerosis, depression, thrombosis and cancers(Horrocks, L. A. and Yeo, Y. K. (1999) Pharmacol. Res. 40:211-215).Therefore, appropriate dietary supply of the fatty acid is important forhuman health. Because such fatty acids cannot be efficiently synthesizedby infants, young children and senior citizerns, it is particularlyimportant for these individuals to adequately intake these fatty acidsfrom the diet (Spector, A. A. (1999) Lipids 34:S1-S3).

Currently the major sources of DHA are oils from fish and algae. Fishoil is a major and traditional source for this fatty acid, however, itis usually oxidized by the time it is sold. In addition, the supply offish oil is highly variable, particularly in view of the shrinking fishpopulations. Moreover, the algal source of oil is expensive due to lowyield and the high costs of extraction.

EPA and ARA are both A5 essential fatty acids. They form a unique classof food and feed constituents for humans and animals. EPA belongs to then-3 series with five double bonds in the acyl chain. EPA is found inmarine food and is abundant in oily fish from North Atlantic. ARAbelongs to the n-6 series with four double bonds. The lack of a doublebond in the ω-3 position confers on ARA different properties than thosefound in EPA. The eicosanoids produced from AA have strong inflammatoryand platelet aggregating properties, whereas those derived from EPA haveanti-inflammatory and anti-platelet aggregating properties. ARA can beobtained from some foods such as meat, fish and eggs, but theconcentration is low.

Gamma-linolenic acid (GLA) is another essential fatty acid found inmammals. GLA is the metabolic intermediate for very long chain n-6 fattyacids and for various active molecules. In mammals, formation of longchain polyunsaturated fatty acids is rate-limited by A6 desaturation.Many physiological and pathological conditions such as aging, stress,diabetes, eczema, and some infections have been shown to depress the A6desaturation step. In addition, GLA is readily catabolized from theoxidation and rapid cell division associated with certain disorders,e.g., cancer or inflammation. Therefore, dietary supplementation withGLA can reduce the risks of these disorders. Clinical studies have shownthat dietary supplementation with GLA is effective in treating somepathological conditions such as atopic eczema, premenstrual syndrome,diabetes, hypercholesterolemia, and inflammatory and cardiovasculardisorders.

A large number of benefitial health effects have been shown for DHA ormixtures of EPA/DHA. DHA is a n-3 very long chain fatty acid with sixdouble bonds.

Although biotechnology offers an attractive route for the production ofspecialty fatty acids, current techniques fail to provide an efficientmeans for the large scale production of unsaturated fatty acids.Accordingly, there exists a need for an improved and efficient method ofproducing unsaturated fatty acids, such as DHA, EPA and ARA.

Thus, the present invention relates to a polynucleotide comprising anucleic acid sequence elected from the group consisting of:

-   a) a nucleic acid sequence having a nucleotide sequence as shown in    SEQ ID NOs: 1, 3, 5, 7 or 9;-   b) a nucleic acid sequence encoding a polypeptide having an amino    acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8 or 10;-   c) a nucleic acid sequence being at least 70% identical to the    nucleic acid sequence of a) or b), wherein said nucleic acid    sequence encodes a polypeptide having desaturase or elongase    activity;-   d) a nucleic acid sequence encoding a polypeptide having desaturase    or elongase activity and having an amino acid sequence which is at    least 82% identical to the amino acid sequence of any one of a) to    c); and-   e) a nucleic acid sequence which is capable of hybridizing under    stringent conditions to any one of a) to d), wherein said nucleic    acid sequence encodes a polypeptide having desaturase or elongase    activity.

The term “polynucleotide” as used in accordance with the presentinvention relates to a polynucleotide comprising a nucleic acid sequencewhich encodes a polypeptide having desaturase or elongase activity.Preferably, the polypeptide encoded by the polynucleotide of the presentinvention having desaturase or elongase activity upon expression in aplant shall be capable of increasing the amount of PUFA and, inparticular, LCPUFA in, e.g., seed oils or the entire plant or partsthereof. Such an increase is, preferably, statistically significant whencompared to a LCPUFA producing transgenic control plant which expressesthe the present state of the art set of desaturases and elongasesrequiered for LCPUFA synthesis but does not express the polynucleotideof the present invention. Whether an increase is significant can bedetermined by statistical tests well known in the art including, e.g.,Student's t-test. More preferably, the increase is an increase of theamount of triglycerides containing LCPUFA of at least 5%, at least 10%,at least 15%, at least 20% or at least 30% compared to the said control.Preferably, the LCPUFA referred to before is a polyunsaturated fattyacid having a C-20 or C-22 fatty acid body, more preferably, ARA, EPA orDHA. Suitable assays for measuring the activities mentioned before aredescribed in the accompanying Examples.

The term “desaturase” or “elongase” as used herein refers to theactivity of a desaturase, introducing a double bond into the carbonchain of a fatty acid, preferably into fatty acids with 18, 20 or 22carbon molecules, or an elongase, introducing two carbon molecules intothe carbon chain of a fatty acid, preferably into fatty acids with 18,20 or 22 carbon molecules

More preferably, polynucleotides having a nucleic acid sequence as shownin SEQ ID NOs: 1, 3, 5, 7 or 9 encoding polypeptides having amino acidsequences as shown in SEQ ID NOs: 2, 4, 6, 8 or 10 or variants thereof,preferably, exhibit desaturase or elongase activity.

Polynucleotides encoding a polypeptide having desaturase or elongaseactivity as specified above has been obtained in accordance with thepresent invention, preferably, from Emiliana huxleyi. However,orthologs, paralogs or other homologs may be identified from otherspecies. Preferably, they are obtained from plants such as algae, forexample Isochrysis, Mantoniella, Ostreococcus or Crypthecodinium,algae/diatoms such as Phaeodactylum, Thalassiosira or Thraustochytrium,mosses such as Physcomitrella or Ceratodon, or higher plants such as thePrimulaceae such as Aleuritia, Calendula stellata, Osteospermumspinescens or Osteospermum hyoseroides, microorganisms such as fungi,such as Aspergillus, Phytophthora, Entomophthora, Mucor or Mortierella,bacteria such as Shewanella, yeasts or animals. Preferred animals arenematodes such as Caenorhabditis, insects or vertebrates. Among thevertebrates, the nucleic acid molecules may, preferably, be derived fromEuteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus, morepreferably, from the order of the Salmoniformes, most preferably, thefamily of the Salmonidae, such as the genus Salmo, for example from thegenera and species Oncorhynchus mykiss, Trutta trutta or Salmo truttafario. Moreover, the nucleic acid molecules may be obtained from thediatoms such as the genera Thallasiosira or Phaeodactylum.

Thus, the term “polynucleotide” as used in accordance with the presentinvention further encompasses variants of the aforementioned specificpolynucleotides representing orthologs, paralogs or other homologs ofthe polynucleotide of the present invention. Moreover, variants of thepolynucleotide of the present invention also include artificiallygenerated muteins. Said muteins include, e.g., enzymes which aregenerated by mutagenesis techniques and which exhibit improved oraltered substrate specificity, or codon optimized polynucleotides. Thepolynucleotide variants, preferably, comprise a nucleic acid sequencecharacterized in that the sequence can be derived from theaforementioned specific nucleic acid sequences shown in any one of SEQID NOs: 1, 3, 5, 7 or 9 or by a polynucleotide encoding a polypeptidehaving an amino acid sequence as shown in any one of SEQ ID NOs: 2, 4,6, 8 or 10 by at least one nucleotide substitution, addition and/ordeletion, whereby the variant nucleic acid sequence shall still encode apolypeptide having a desaturase or elongase activity as specified above.Variants also encompass polynucleotides comprising a nucleic acidsequence which is capable of hybridizing to the aforementioned specificnucleic acid sequences, preferably, under stringent hybridizationconditions. These stringent conditions are known to the skilled workerand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred example for stringenthybridization conditions are hybridization conditions in 6×sodiumchloride/sodium citrate (=SSC) at approximately 45° C., followed by oneor more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. The skilledworker knows that these hybridization conditions differ depending on thetype of nucleic acid and, for example when organic solvents are present,with regard to the temperature and concentration of the buffer. Forexample, under “standard hybridization conditions” the temperaturediffers depending on the type of nucleic acid between 42° C. and 58° C.in aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). Iforganic solvent is present in the abovementioned buffer, for example 50%formamide, the temperature under standard conditions is approximately42° C. The hybridization conditions for DNA: DNA hybrids are,preferably, 0.1×SSC and 20° C. to 45° C., preferably between 30° C. and45° C. The hybridization conditions for DNA:RNA hybrids are, preferably,0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55° C. Theabovementioned hybridization temperatures are determined for example fora nucleic acid with approximately 100 bp (=base pairs) in length and aG+C content of 50% in the absence of formamide. The skilled worker knowshow to determine the hybridization conditions required by referring totextbooks such as the textbook mentioned above, or the followingtextbooks: Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer-based amplification ofDNA, i.e. using degenerated primers against conserved domains of thepolypeptides of the present invention. Conserved domains of thepolypeptide of the present invention may be identified by a sequencecomparison of the nucleic acid sequences of the polynucleotides or theamino acid sequences of the polypeptides of the present invention.Oligonucleotides suitable as PCR primers as well as suitable PCRconditions are described in the accompanying Examples. As a template,DNA or cDNA from bacteria, fungi, plants or animals may be used.Further, variants include polynucleotides comprising nucleic acidsequences which are at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or at least 99% identical to the nucleicacid sequences shown in any one of SEQ ID NOs: 1, 3, 5, 7 or 9,preferably, encoding polypeptides retaining a desaturase or elongaseactivity as specified above. Moreover, also encompassed arepolynucleotides which comprise nucleic acid sequences encoding apolypeptide having an amino acid sequences which are at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% identical to the amino acid sequences shown in any one of SEQID NOs: 2, 4, 6, 8 or 10, wherein the polypeptide, preferably, retainsdesaturase or elongase activity as specified above. The percent identityvalues are, preferably, calculated over the entire amino acid or nucleicacid sequence region. A series of programs based on a variety ofalgorithms is available to the skilled worker for comparing differentsequences. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunschalgorithm (Needleman 1970, J. Mol. Biol. (48):444-453) which has beenincorporated into the needle program in the EMBOSS software package(EMBOSS: The European Molecular Biology Open Software Suite, Rice, P.,Longden, I., and Bleasby, A, Trends in Genetics 16(6), 276-277, 2000),using either a BLOSUM 45 or PAM250 scoring matrix for distantly relatedproteins, or either a BLOSUM 62 or PAM160 scoring matrix for closerrelated proteins, and a gap opening penalty of 16, 14, 12, 10, 8, 6, or4 and a gap entension pentalty of 0.5, 1, 2, 3, 4, 5, or 6. Guides forlocal installation of the EMBOSS package as well as links toWEB-Services can be found at emboss.sourceforge.net. A preferred,non-limiting example of parameters to be used for aligning two aminoacid sequences using the needle program are the default parameters,including the EBLOSUM62 scoring matrix, a gap opening penalty of 10 anda gap extension penalty of 0.5. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe needle program in the EMBOSS software package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice, P., Longden, I., andBleasby, A, Trends in Genetics 16(6), 276-277, 2000), using the EDNAFULLscoring matrix and a gap opening penalty of 16, 14, 12, 10, 8, 6, or 4and a gap extension penalty of 0.5, 1, 2, 3, 4, 5, or 6. A preferred,non-limiting example of parameters to be used in conjunction foraligning two amino acid sequences using the needle program are thedefault parameters, including the EDNAFULL scoring matrix, a gap openingpenalty of 10 and a gap extension penalty of 0.5. The nucleic acid andprotein sequences of the present invention can further be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the BLAST series of programs (version2.2) of Altschul et al. (Altschul 1990, J. Mol. Biol. 215:403-10). BLASTusing acyltransferase nucleic acid sequences of the invention as querysequence can be performed with the BLASTn, BLASTx or the tBLASTx programusing default parameters to obtain either nucleotide sequences (BLASTn,tBLASTx) or amino acid sequences (BLASTx) homologous to acyltransferasesequences of the invention. BLAST using acyltransferase proteinsequences of the invention as query sequence can be performed with theBLASTp or the tBLASTn program using default parameters to obtain eitheramino acid sequences (BLASTp) or nucleic acid sequences (tBLASTn)homologous to acyltransferase sequences of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST using defaultparameters can be utilized as described in Altschul et al. (Altschul1997, Nucleic Acids Res. 25(17):3389-3402).

TABLE 1 Relation of sequence types of querry and hit sequences forvarious BLAST programs Input Converted Converted Actual query sequenceQuery Algorithm Hit Database DNA BLASTn DNA PRT BLASTp PRT DNA PRTBLASTx PRT PRT tBLASTn PRT DNA DNA PRT tBLASTx PRT DNA

A polynucleotide comprising a fragment of any of the aforementionednucleic acid sequences is also encompassed as a polynucleotide of thepresent invention. The fragment shall encode a polypeptide which stillhas desaturase and elongase activity as specified above. Accordingly,the polypeptide may comprise or consist of the domains of thepolypeptide of the present invention conferring the said biologicalactivity. A fragment as meant herein, preferably, comprises at least 50,at least 100, at least 250 or at least 500 consecutive nucleotides ofany one of the aforementioned nucleic acid sequences or encodes an aminoacid sequence comprising at least 20, at least 30, at least 50, at least80, at least 100 or at least 150 consecutive amino acids of any one ofthe aforementioned amino acid sequences.

The variant polynucleotides or fragments referred to above, preferably,encode polypeptides retaining desaturase or elongase activity to asignificant extent, preferably, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% of the desaturase and elongase activity exhibited byany of the polypeptide shown in any one of SEQ ID NOs: 2, 4, 6, 8 or 10.The activity may be tested as described in the accompanying Examples.

The polynucleotides of the present invention either essentially consistof the aforementioned nucleic acid sequences or comprise theaforementioned nucleic acid sequences. Thus, they may contain furthernucleic acid sequences as well. Preferably, the polynucleotide of thepresent invention may comprise in addition to an open reading framefurther untranslated sequence at the 3′ and at the 5′ terminus of thecoding gene region: at least 500, preferably 200, more preferably 100nucleotides of the sequence upstream of the 5′ terminus of the codingregion and at least 100, preferably 50, more preferably 20 nucleotidesof the sequence downstream of the 3′ terminus of the coding gene region.Furthermore, the polynucleotides of the present invention may encodefusion proteins wherein one partner of the fusion protein is apolypeptide being encoded by a nucleic acid sequence recited above. Suchfusion proteins may comprise as additional part other enzymes of thefatty acid or PUFA biosynthesis pathways, polypeptides for monitoringexpression (e.g., green, yellow, blue or red fluorescent proteins,alkaline phosphatase and the like) or so called “tags” which may serveas a detectable marker or as an auxiliary measure for purificationpurposes. Tags for the different purposes are well known in the art andcomprise FLAG-tags, 6-histidine-tags, MYC-tags and the like.

The polynucleotide of the present invention shall be provided,preferably, either as an isolated polynucleotide (i.e. purified or atleast isolated from its natural context such as its natural gene locus)or in genetically modified or exogenously (i.e. artificially)manipulated form. An isolated polynucleotide can, for example, compriseless than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleotide sequences which naturally flank the nucleic acid moleculein the genomic DNA of the cell from which the nucleic acid is derived.The polynucleotide, preferably, is provided in the form of double orsingle stranded molecule. It will be understood that the presentinvention by referring to any of the aforementioned polynucleotides ofthe invention also refers to complementary or reverse complementarystrands of the specific sequences or variants thereof referred tobefore. The polynucleotide encompasses DNA, including cDNA and genomicDNA, or RNA polynucleotides.

However, the present invention also pertains to polynucleotide variantswhich are derived from the polynucleotides of the present invention andare capable of interefering with the transcription or translation of thepolynucleotides of the present invention. Such variant polynucleotidesinclude anti-sense nucleic acids, ribozymes, siRNA molecules, morpholinonucleic acids (phosphorodiamidate morpholino oligos), triple-helixforming oligonucleotides, inhibitory oligonucleotides, or micro RNAmolecules all of which shall specifically recognize the polynucleotideof the invention due to the presence of complementary or substantiallycomplementary sequences. These techniques are well known to the skilledartisan. Suitable variant polynucleotides of the aforementioned kind canbe readily designed based on the structure of the polynucleotides ofthis invention.

Moreover, comprised are also chemically modified polynucleotidesincluding naturally occurring modified polynucleotides such asglycosylated or methylated polynucleotides or artificial modified onessuch as biotinylated polynucleotides.

In the studies underlying the present invention, advantageously,polynucleotides where identified encoding desaturase and elongases fromEmiliana huxleyi. In particular, the Emiliana huxleyi desaturasesΔ4Des(Eh), Δ8Des(Eh) and Δ5Des(Eh) and elongases Δ9Elo(Eh) and Δ5Elo(Eh)have been identified. Each of the desaturases are capable of introducinga double bond into fatty acids. For example, the expression of theΔ8Des(Eh) leads to introduction of a double bond at position eight intoC20:2n-6 fatty acid. The polynucleotides of the present invention areparticularly suitable for the recombinant manufacture of LCPUFAs and, inparticular, ARA, EPA and/or DHA.

In a preferred embodiment of the polynucleotide of the presentinvention, said polynucleotide further comprises an expression controlsequence operatively linked to the said nucleic acid sequence.

The term “expression control sequence” as used herein refers to anucleic acid sequence which is capable of governing, i.e. initiating andcontrolling, transcription of a nucleic acid sequence of interest, inthe present case the nucleic sequences recited above. Such a sequenceusually comprises or consists of a promoter or a combination of apromoter and enhancer sequences. Expression of a polynucleotidecomprises transcription of the nucleic acid molecule, preferably, into atranslatable mRNA. Additional regulatory elements may includetranscriptional as well as translational enhancers. The followingpromoters and expression control sequences may be, preferably, used inan expression vector according to the present invention. The cos, tac,trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara,SP6, λ-PR or λ-PL promoters are, preferably, used in Gram-negativebacteria. For Gram-positive bacteria, promoters amy and SPO2 may beused. From yeast or fungal promoters ADC1, AOX1r, GAL1, MFα, AC, P-60,CYC1, GAPDH, TEF, rp28, ADH are, preferably, used. For animal cell ororganism expression, the promoters CMV-, SV40-, RSV-promoter (Roussarcoma virus), CMV-enhancer, SV40-enhancer are preferably used. Fromplants the promoters CaMV/35S (Franck 1980, Cell 21: 285-294], PRP1(Ward 1993, Plant. Mol. Biol. 22), SSU, OCS, lib4, usp, STLS1, B33, nosor the ubiquitin or phaseolin promoter. Also preferred in this contextare inducible promoters, such as the promoters described in EP 0 388 186A1 (i.e. a benzylsulfonamide-inducible promoter), Gatz 1992, Plant J.2:397-404 (i.e. a tetracyclin-inducible promoter), EP 0 335 528 A1 (i.e.a abscisic-acid-inducible promoter) or WO 93/21334 (i.e. a ethanol- orcyclohexenol-inducible promoter). Further suitable plant promoters arethe promoter of cytosolic FBPase or the ST-LSI promoter from potato(Stockhaus 1989, EMBO J. 8, 2445), the phosphoribosyl-pyrophosphateamidotransferase promoter from Glycine max (Genbank accession No.U87999) or the node-specific promoter described in EP 0 249 676 Δ1.Particularly preferred are promoters which enable the expression intissues which are involved in the biosynthesis of fatty acids. Alsoparticularly preferred are seed-specific promoters such as the USPpromoter in accordance with the practice, but also other promoters suchas the LeB4, DC3, phaseolin or napin promoters. Further especiallypreferred promoters are seed-specific promoters which can be used formonocotyledonous or dicotyledonous plants and which are described inU.S. Pat. No. 5,608,152 (napin promoter from oilseed rape), WO 98/45461(oleosin promoter from Arobidopsis, U.S. Pat. No. 5,504,200 (phaseolinpromoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter fromBrassica), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots. Thefollowing promoters are suitable for monocots: lpt-2 or lpt-1 promoterfrom barley (WO 95/15389 and WO 95/23230), hordein promoter from barleyand other promoters which are suitable and which are described in WO99/16890. In principle, it is possible to use all natural promoterstogether with their regulatory sequences, such as those mentioned above,for the novel process. Likewise, it is possible and advantageous to usesynthetic promoters, either additionally or alone, especially when theymediate a seed-specific expression, such as, for example, as describedin WO 99/16890. In a particular embodiment, seed-specific promoters areutilized to enhance the production of the desired PUFA or LCPUFA.

The term “operatively linked” as used herein means that the expressioncontrol sequence and the nucleic acid of interest are linked so that theexpression of the said nucleic acid of interest can be governed by thesaid expression control sequence, i.e. the expression control sequenceshall be functionally linked to the said nucleic acid sequence to beexpressed. Accordingly, the expression control sequence and, the nucleicacid sequence to be expressed may be physically linked to each other,e.g., by inserting the expression control sequence at the 5″end of thenucleic acid sequence to be expressed. Alternatively, the expressioncontrol sequence and the nucleic acid to be expressed may be merely inphysical proximity so that the expression control sequence is capable ofgoverning the expression of at least one nucleic acid sequence ofinterest. The expression control sequence and the nucleic acid to beexpressed are, preferably, separated by not more than 500 bp, 300 bp,100 bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 bp or 5 bp.

In a further preferred embodiment of the polynucleotide of the presentinvention, said polynucleotide further comprises a terminator sequenceoperatively linked to the nucleic acid sequence.

The term “terminator” as used herein refers to a nucleic acid sequencewhich is capable of terminating transcription. These sequences willcause dissociation of the transcription machinery from the nucleic acidsequence to be transcribed. Preferably, the terminator shall be activein plants and, in particular, in plant seeds. Suitable terminators areknown in the art and, preferably, include polyadenylation signals suchas the SV40-poly-A site or the tk-poly-A site or one of the plantspecific signals indicated in Loke et al. (Loke 2005, Plant Physiol 138,pp. 1457-1468), downstream of the nucleic acid sequence to be expressed.

The present invention also relates to a vector comprising thepolynucleotide of the present invention.

The term “vector”, preferably, encompasses phage, plasmid, viral vectorsas well as artificial chromosomes, such as bacterial or yeast artificialchromosomes. Moreover, the term also relates to targeting constructswhich allow for random or site-directed integration of the targetingconstruct into genomic DNA. Such target constructs, preferably, compriseDNA of sufficient length for either homolgous or heterologousrecombination as described in detail below. The vector encompassing thepolynucleotide of the present invention, preferably, further comprisesselectable markers for propagation and/or selection in a host. Thevector may be incorporated into a host cell by various techniques wellknown in the art. If introduced into a host cell, the vector may residein the cytoplasm or may be incorporated into the genome. In the lattercase, it is to be understood that the vector may further comprisenucleic acid sequences which allow for homologous recombination orheterologous insertion. Vectors can be introduced into prokaryotic oreukaryotic cells via conventional transformation or transfectiontechniques. The terms “transformation” and “transfection”, conjugationand transduction, as used in the present context, are intended tocomprise a multiplicity of prior-art processes for introducing foreignnucleic acid (for example DNA) into a host cell, including calciumphosphate, rubidium chloride or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,carbon-based clusters, chemically mediated transfer, electroporation orparticle bombardment. Suitable methods for the transformation ortransfection of host cells, including plant cells, can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) and other laboratory manuals, such as Methodsin Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.:Gartland and Davey, Humana Press, Totowa, N.J. Alternatively, a plasmidvector may be introduced by heat shock or electroporation techniques.Should the vector be a virus, it may be packaged in vitro using anappropriate packaging cell line prior to application to host cells.

Preferably, the vector referred to herein is suitable as a cloningvector, i.e. replicable in microbial systems. Such vectors ensureefficient cloning in bacteria and, preferably, yeasts or fungi and makepossible the stable transformation of plants. Those which must bementioned are, in particular, various binary and co-integrated vectorsystems which are suitable for the T-DNA-mediated transformation. Suchvector systems are, as a rule, characterized in that they contain atleast the vir genes, which are required for the Agrobacterium-mediatedtransformation, and the sequences which delimit the T-DNA (T-DNAborder). These vector systems, preferably, also comprise furthercis-regulatory regions such as promoters and terminators and/orselection markers with which suitable transformed host cells ororganisms can be identified. While co-integrated vector systems have virgenes and T-DNA sequences arranged on the same vector, binary systemsare based on at least two vectors, one of which bears vir genes, but noT-DNA, while a second one bears T-DNA, but no vir gene. As aconsequence, the last-mentioned vectors are relatively small, easy tomanipulate and can be replicated both in E. coli and in Agrobacterium.These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks,pGreen series. Preferably used in accordance with the invention areBin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview of binary vectorsand their use can be found in Hellens et al, Trends in Plant Science(2000) 5, 446-451. Furthermore, by using appropriate cloning vectors,the polynucleotides can be introduced into host cells or organisms suchas plants or animals and, thus, be used in the transformation of plants,such as those which are published, and cited, in: Plant MolecularBiology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7,pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in HigherPlants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.:Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniquesfor Gene Transfer, in: Transgenic Plants, vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143;Potrykus 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205-225.

More preferably, the vector of the present invention is an expressionvector. In such an expression vector, i.e. a vector which comprises thepolynucleotide of the invention having the nucleic acid sequenceoperatively linked to an expression control sequence (also called“expression cassette”) allowing expression in prokaryotic or eukaryoticcells or isolated fractions thereof. Suitable expression vectors areknown in the art such as Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogene) or pSPORT1(GIBCO BRL). Further examples of typical fusion expression vectors arepGEX (Pharmacia Biotech Inc; Smith 1988, Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,N.J.), where glutathione S-transferase (GST), maltose E-binding proteinand protein A, respectively, are fused with the recombinant targetprotein. Examples of suitable inducible nonfusion E. coli expressionvectors are, inter alia, pTrc (Amann 1988, Gene 69:301-315) and pET 11d(Studier 1990, Methods in Enzymology 185, 60-89). The target geneexpression of the pTrc vector is based on the transcription from ahybrid trp-lac fusion promoter by host RNA polymerase. The target geneexpression from the pET 11d vector is based on the transcription of aT7-gn10-lac fusion promoter, which is mediated by a coexpressed viralRNA polymerase (T7 gn1). This viral polymerase is provided by the hoststrains BL21 (DE3) or HMS174 (DE3) from a resident λ-prophage whichharbors a T7 gn1 gene under the transcriptional control of the lacUV 5promoter. The skilled worker is familiar with other vectors which aresuitable in prokaryotic organisms; these vectors are, for example, in E.coli, pLG338, pACYC184, the pBR series such as pBR322, the pUC seriessuch as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2,pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCl, inStreptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194or pBD214, in Corynebacterium pSA77 or pAJ667. Examples of vectors forexpression in the yeast S. cerevisiae comprise pYep Sec1 (Baldari 1987,Embo J. 6:229-234), pMFa (Kurjan 1982, Cell 30:933-943), pJRY88 (Schultz1987, Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and processes for the construction of vectors which aresuitable for use in other fungi, such as the filamentous fungi, comprisethose which are described in detail in: van den Hondel, C. A. M. J. J.,& Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of fungi, J. F.Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, orin: More Gene Manipulations in Fungi O. W. Bennett & L. L. Lasure, Ed.,pp. 396-428: Academic Press: San Diego). Further suitable yeast vectorsare, for example, pAG-1, YEp6, YEp13 or pEMBLYe23. As an alternative,the polynucleotides of the present invention can be also expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorswhich are available for the expression of proteins in cultured insectcells (for example Sf9 cells) comprise the pAc series (Smith 1983, Mol.Cell Biol. 3:2156-2165) and the pVL series (Lucklow 1989, Virology170:31-39).

The polynucleotide of the present invention can be expressed insingle-cell plant cells (such as algae), see Falciatore 1999, MarineBiotechnology 1 (3):239-251 and the references cited therein, and plantcells from higher plants (for example Spermatophytes, such as arablecrops) by using plant expression vectors. Examples of plant expressionvectors comprise those which are described in detail in: Becker 1992,Plant Mol. Biol. 20:1195-1197; Bevan 1984, Nucl. Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu,Academic Press, 1993, p. 15-38. A plant expression cassette, preferably,comprises regulatory sequences which are capable of controlling the geneexpression in plant cells and which are functionally linked so that eachsequence can fulfill its function, such as transcriptional termination,for example polyadenylation signals. Preferred polyadenylation signalsare those which are derived from Agrobacterium tumefaciens T-DNA, suchas the gene 3 of the Ti plasmid pTiACH5, which is known as octopinesynthase (Gielen 1984, EMBO J. 3, 835) or functional equivalents ofthese, but all other terminators which are functionally active in plantsare also suitable. Since plant gene expression is very often not limitedto transcriptional levels, a plant expression cassette preferablycomprises other functionally linked sequences such as translationenhancers, for example the overdrive sequence, which comprises the5′-untranslated tobacco mosaic virus leader sequence, which increasesthe protein/RNA ratio (Gallie 1987, Nucl. Acids Research 15:8693-8711).As described above, plant gene expression must be functionally linked toa suitable promoter which performs the expression of the gene in atimely, cell-specific or tissue-specific manner. Promoters which can beused are constitutive promoters (Benfey 1989, EMBO J. 8:2195-2202) suchas those which are derived from plant viruses such as 35S CAMV (Franck1980, Cell 21:285-294), 19S CaMV (see U.S. Pat. No. 5,352,605 and WO84/02913) or plant promoters such as the promoter of the Rubisco smallsubunit, which is described in U.S. Pat. No. 4,962,028. Other preferredsequences for the use in functional linkage in plant gene expressioncassettes are targeting sequences which are required for targeting thegene product into its relevant cell compartment (for a review, seeKermode 1996, Crit. Rev. Plant Sci. 15, 4: 285-423 and references citedtherein), for example into the vacuole, the nucleus, all types ofplastids, such as amyloplasts, chloroplasts, chromoplasts, theextracellular space, the mitochondria, the endoplasmic reticulum, oilbodies, peroxisomes and other compartments of plant cells. As describedabove, plant gene expression can also be facilitated via a chemicallyinducible promoter (for a review, see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108). Chemically inducible promotersare particularly suitable if it is desired that genes are expressed in atime-specific manner. Examples of such promoters are asalicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-induciblepromoter (Gatz 1992, Plant J. 2, 397-404) and an ethanol-induciblepromoter. Promoters which respond to biotic or abiotic stress conditionsare also suitable promoters, for example the pathogen-induced PRP1-genepromoter (Ward 1993, Plant Mol. Biol. 22:361-366), the heat-induciblehsp80 promoter from tomato (U.S. Pat. No. 5,187,267), the cold-induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII promoter (EP 0 375 091 A). The promoters which are especiallypreferred are those which bring about the expression of genes in tissuesand organs in which fatty acid, lipid and oil biosynthesis takes place,in seed cells such as the cells of endosperm and of the developingembryo. Suitable promoters are the napin gene promoter from oilseed rape(U.S. Pat. No. 5,608,152), the USP promoter from Vicia faba (Baeumlein1991, Mol. Gen. Genet. 225 (3):459-67), the oleosin promoter fromArabidopsis (WO 98/45461), the phaseolin promoter from Phaseolusvulgaris (U.S. Pat. No. 5,504,200), the Bce4 promoter from Brassica (WO91/13980) or the legumin B4 promoter (LeB4; Baeumlein 1992, PlantJournal, 2 (2):233-9), and promoters which bring about the seed-specificexpression in monocotyledonous plants such as maize, barley, wheat, rye,rice and the like. Suitable promoters to be taken into consideration arethe lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230)or those which are described in WO 99/16890 (promoters from the barleyhordein gene, the rice glutelin gene, the rice oryzin gene, the riceprolamin gene, the wheat gliadin gene, wheat glutelin gene, the maizezein gene, the oat glutelin gene, the sorghum kasirin gene, the ryesecalin gene). Likewise, especially suitable are promoters which bringabout the plastid-specific expression since plastids are the compartmentin which the precursors and some end products of lipid biosynthesis aresynthesized. Suitable promoters such as the viral RNA-polymerasepromoter, are described in WO 95/16783 and WO 97/06250, and the clpPpromoter from Arabidopsis, described in WO 99/46394.

The abovementioned vectors are only a small overview of vectors to beused in accordance with the present invention. Further vectors are knownto the skilled worker and are described, for example, in: CloningVectors (Ed., Pouwels, P. H., et al., Elsevier, Amsterdam-NewYork-Oxford, 1985, ISBN 0 444 904018). For further suitable expressionsystems for prokaryotic and eukaryotic cells see the chapters 16 and 17of Sambrook, loc cit.

It follows from the above that, preferably, said vector is an expressionvector. More preferably, the said polynucleotide of the presentinvention is under the control of a seed-specific promoter in the vectorof the present invention. A preferred seed-specific promoter as meantherein is selected from the group consisting of Conlinin 1, Conlinin 2,napin, LuFad3, USP, LeB4, Arc, Fae, ACP, LuPXR, and SBP. For details,see, e.g., US 2003-0159174.

Moreover, the present invention relates to a host cell comprising thepolynucleotide or the vector of the present invention.

Preferably, said host cell is a plant cell and, more preferably, a plantcell obtained from an oilseed crop. More preferably, said oilseed cropis selected from the group consisting of flax (Linum sp.), rapeseed(Brassica sp.), soybean (Glycine sp.), sunflower (Helianthus sp.),cotton (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower(Carthamus sp.), cocoa (Theobroma cacoa), peanut (Arachissp.), hemp,camelina, crambe, oil palm, coconuts, groundnuts, sesame seed, castorbean, lesquerella, tallow tree, sheanuts, tungnuts, kapok fruit, poppyseed, jojoba seeds and perilla.

Also preferably, said host cell is a microorganism. More preferably,said microorganism is a bacterium, a fungus or algae. More preferably,it is selected from the group consisting of Candida, Cryptococcus,Lipomyces, Rhodosporidium, Yarrowia and Schizochytrium.

Moreover, a host cell according to the present invention may also be ananimal cell. Preferably, said animal host cell is a host cell of a fishor a cell line obtained therefrom. More preferably, the fish host cellis from herring, salmon, sardine, redfish, eel, carp, trout, halibut,mackerel, zander or tuna.

Generally, the controlling steps in the production of LCPUFAs, i.e., thelong chain unsaturated fatty acid biosynthetic pathway, are catalyzed bymembrane-associated fatty acid desaturases and elongases. Plants andmost other eukaryotic organisms have specialized desaturase and elongasesystems for the introduction of double bonds and the extension of fattyacids beyond C18 atoms. The elongase reactions have several importantfeatures in common with the fatty acid synthase complex (FAS). However,the elongase complex is different from the FAS complex as the complex islocalized in the cytosol and membrane bound, ACP is not involved and theelongase 3-keto-acyl-CoA-synthase catalyzes the condensation ofmalonyl-CoA with an acyl primer. The elongase complex consists of fourcomponents with different catalytic functions, the keto-acyl-synthase(condensation reaction of malonyl-CoA to acyl-CoA, creation of a 2 Catom longer keto-acyl-CoA fatty acid), the keto-acyl-reductase(reduction of the 3-keto group to a 3-hydroxy-group), the dehydratase(dehydration results in a 3-enoyl-acyl-CoA fatty acid) and theenoly-CoA-reductase (reduction of the double bond at position 3, releasefrom the complex). For the production of LCPUFAs including ARA, EPAand/or DHA the elongation reactions, beside the desaturation reactions,are essential. Higher plants do not have the necessary enzyme set toproduce LCPUFAs (4 or more double bonds, 20 or more C atoms). Thereforethe catalytic activities have to be conferred to the plants or plantcells. The polynucleotides of the present invention catalyze thedesaturation and elongation activities necessary for the formation ofARA, EPA and/or DHA. By delivering the novel desaturases and elongasesincreased levels of PUFAs and LCPUFAs are produced.

However, person skilled in the art knows that dependent on the hostcell, further, enzymatic activities may be conferred to the host cells,e.g., by recombinant technologies. Accordingly, the present invention,preferably, envisages a host cell which in addition to thepolynucleotide of the present invention comprises polynucleotidesencoding such desaturases and/or elongases as required depending on theselected host cell. Preferred desaturases and/or elongases which shallbe present in the host cell are at least one enzyme selected from thegroup consisting of: Δ-4-desaturase, Δ-5-desaturase, Δ-5-elongase,Δ-6-desaturase, Δ12-desaturase, Δ15-desaturase, ω3-desaturase andΔ-6-elongase. Especially prefered are the bifunctionald12d15-Desaturases d12d15Des(Ac) from Acanthamoeba castellanii(WO2007042510), d12d15Des(Cp) from Claviceps purpurea (WO2008006202) andd12d15Des(Lg)1 from Lottia gigantea (WO2009016202), the d12-Desaturasesd12Des(Co) from Calendula officinalis (WO200185968), d12Des(Lb) fromLaccaria bicolor (WO2009016202), d12Des(Mb) from Monosiga brevicollis(WO2009016202), d12Des(Mg) from Mycosphaerella graminicola(WO2009016202), d12Des(Nh) from Nectria haematococca (WO2009016202),d12Des(Ol) from Ostreococcus lucimarinus (WO2008040787), d12Des(Pb) fromPhycomyces blakesleeanus (WO2009016202), d12Des(Ps) from Phytophthorasojae (WO2006100241) and d12Des(Tp) from Thalassiosira pseudonana(WO2006069710), the d15-Desaturases d15Des(Hr) from Helobdella robusta(WO2009016202), d15Des(Mc) from Microcoleus chthonoplastes(WO2009016202), d15Des(Mf) from Mycosphaerella fijiensis (WO2009016202),d15Des(Mg) from Mycosphaerella graminicola (WO2009016202) andd15Des(Nh)2 from Nectria haematococca (WO2009016202), the d4-Desaturasesd4Des(Eg) from Euglena gracilis (WO2004090123), d4Des(Tc) fromThraustochytrium sp. (WO2002026946) and d4Des(Tp) from Thalassiosirapseudonana (WO2006069710), the d5-Desaturases d5Des(Ol)2 fromOstreococcus lucimarinus (WO2008040787), d5Des(Pp) from Physcomitrellapatens (WO2004057001), d5Des(Pt) from Phaeodactylum tricornutum(WO2002057465), d5Des(Tc) from Thraustochytrium sp. (WO2002026946),d5Des(Tp) from Thalassiosira pseudonana (WO2006069710) and thed6-Desaturases d6Des(Cp) from Ceratodon purpureus (WO2000075341),d6Des(Ol) from Ostreococcus lucimarinus (WO2008040787), d6Des(Ot) fromOstreococcus tauri (WO2006069710), d6Des(Pf) from Primula farinosa(WO2003072784), d6Des(Pir)_BO from Pythium irregulare (WO2002026946),d6Des(Pir) from Pythium irregulare (WO2002026946), d6Des(Plu) fromPrimula luteola (WO2003072784), d6Des(Pp) from Physcomitrella patens(WO200102591), d6Des(Pt) from Phaeodactylum tricornutum (WO2002057465),d6Des(Pv) from Primula vialii (WO2003072784) and d6Des(Tp) fromThalassiosira pseudonana (WO2006069710), the d8-Desaturases d8Des(Ac)from Acanthamoeba castellanii (EP1790731), d8Des(Eg) from Euglenagracilis (WO200034439) and d8Des(Pm) from Perkinsus marinus(WO2007093776), the o3-Desaturases o3Des(Pi) from Phytophthora infestans(WO2005083053), o3Des(Pir) from Pythium irregulare (WO2008022963),o3Des(Pir)2 from Pythium irregulare (WO2008022963) and o3Des(Ps) fromPhytophthora sojae (WO2006100241), the bifunctional d5d6-elongasesd5d6Elo(Om)2 from Oncorhynchus mykiss (WO2005012316), d5d6Elo(Ta) fromThraustochytrium aureum (WO2005012316) and d5d6Elo(Tc) fromThraustochytrium sp. (WO2005012316), the d5-elongases d5Elo(At) fromArabidopsis thaliana (WO2005012316), d5Elo(At)2 from Arabidopsisthaliana (WO2005012316), d5Elo(Ci) from Ciona intestinalis(WO2005012316), d5Elo(Ol) from Ostreococcus lucimarinus (WO2008040787),d5Elo(Ot) from Ostreococcus tauri (WO2005012316), d5Elo(Tp) fromThalassiosira pseudonana (WO2005012316) and d5Elo(XI) from Xenopuslaevis (WO2005012316), the d6-elongases d6Elo(Ol) from Ostreococcuslucimarinus (WO2008040787), d6Elo(Ot) from Ostreococcus tauri(WO2005012316), d6Elo(Pi) from Phytophthora infestans (WO2003064638),d6Elo(Pir) from Pythium irregulare (WO2009016208), d6Elo(Pp) fromPhyscomitrella patens (WO2001059128), d6Elo(Ps) from Phytophthora sojae(WO2006100241), d6Elo(Ps)2 from Phytophthora sojae (WO2006100241),d6Elo(Ps)3 from Phytophthora sojae (WO2006100241), d6Elo(Pt) fromPhaeodactylum tricornutum (WO2005012316), d6Elo(Tc) fromThraustochytrium sp. (WO2005012316) and d6Elo(Tp) from Thalassiosirapseudonana (WO2005012316), the d9-elongases d9Elo(Ig) from Isochrysisgalbana (WO2002077213), d9Elo(Pm) from Perkinsus marinus (WO2007093776)and d9Elo(Ro) from Rhizopus oryzae (WO2009016208). Particularly, if themanufuacture of ARA is envisaged in higher plants, the enzymes recitedin Table 3, below (i.e. aditinonally a d6-desaturase, d6-elongase,d5-elongase, d5-desaturase, d12-desaturase, and d6-elongase) or enzymeshaving essentially the same activity may be combined in a host cell. Ifthe manufacture of EPA is envisaged in higher plants, the enzymesrecited in Table 4, below (i.e. aditinonally a d6-desaturase,d6-elongase, d5-desaturase, d12-desaturase, d6-elongase, omega3-desaturase and d15-desaturase), or enzymes having essentially the sameactivity may be combined in a host cell. If the manufacture of DHA isenvisaged in higher plants, the enzymes recited in Table 5, below (i.e.aditinonally a d6-desaturase, d6-elongase, d5-desaturase,d12-desaturase, d6-elongase, omega 3-desaturase, d15-desaturase,d5-elongase, and d4-desaturase), or enzymes having essentially the sameactivity may be combined in a host cell.

The present invention also relates to a cell, preferably a host cell asspecified above or a cell of a non-human organism specified elsewhereherein, said cell comprising a polynucleotide which is obtained from thepolynucleotide of the present invention by a point mutation, atruncation, an inversion, a deletion, an addition, a substitution andhomologous recombination. How to carry out such modifications to apolynucleotide is well known to the skilled artisan and has beendescribed elsewhere in this specification in detail.

The present invention furthermore pertains to a method for themanufacture of a polypeptide encoded by a polynucleotide of any thepresent invention comprising

-   a) cultivating the host cell of the invention under conditions which    allow for the production of the said polypeptide; and-   b) obtaining the polypeptide from the host cell of step a).

Suitable conditions which allow for expression of the polynucleotide ofthe invention comprised by the host cell depend on the host cell as wellas the expression control sequence used for governing expression of thesaid polynucleotide. These conditions and how to select them are verywell known to those skilled in the art. The expressed polypeptide may beobtained, for example, by all conventional purification techniquesincluding affinity chromatography, size exclusion chromatography, highpressure liquid chromatography (HPLC) and precipitation techniquesincluding antibody precipitation. It is to be understood that the methodmay—although preferred—not necessarily yield an essentially purepreparation of the polypeptide. It is to be understood that depending onthe host cell which is used for the aforementioned method, thepolypeptides produced thereby may become posttranslationally modified orprocessed otherwise.

The present invention also encompasses a polypeptide encoded by thepolynucleotide of of the present invention or which is obtainable by theaforementioned method.

The term “polypeptide” as used herein encompasses essentially purifiedpolypeptides or polypeptide preparations comprising other proteins inaddition. Further, the term also relates to the fusion proteins orpolypeptide fragments being at least partially encoded by thepolynucleotide of the present invention referred to above. Moreover, itincludes chemically modified polypeptides. Such modifications may beartificial modifications or naturally occurring modifications such asphosphorylation, glycosylation, myristylation and the like (Review inMann 2003, Nat. Biotechnol. 21, 255-261, review with focus on plants inHuber 2004, Curr. Opin. Plant Biol. 7, 318-322). Currently, more than300 posttranslational modifications are known (see full ABFRC Delta masslist at abrf.org/index.cfm/dm.home). The polypeptides of the presentinvention shall exhibit the desaturase or elongase activitiy referred toabove.

Encompassed by the present invention is, furthermore, an antibody orfragments thereof which specifically recognizes the polypeptide of theinvention.

Antibodies against the polypeptides of the invention can be prepared bywell known methods using a purified polypeptide according to theinvention or a suitable fragment derived therefrom as an antigen. Afragment which is suitable as an antigen may be identified byantigenicity determining algorithms well known in the art. Suchfragments may be obtained either from the polypeptide of the inventionby proteolytic digestion or may be a synthetic peptide. Preferably, theantibody of the present invention is a monoclonal antibody, a polyclonalantibody, a single chain antibody, a chimerized antibody or a fragmentof any of these antibodies, such as Fab, Fv or scFv fragments etc. Alsocomprised as antibodies by the present invention are bispecificantibodies, synthetic antibodies or chemically modified derivatives ofany of the aforementioned antibodies. The antibody of the presentinvention shall specifically bind (i.e. does significantly not crossreact with other polypeptides or peptides) to the polypeptide of theinvention. Specific binding can be tested by various well knowntechniques. Antibodies or fragments thereof can be obtained by usingmethods which are described, e.g., in Harlow and Lane “Antibodies, ALaboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Monoclonalantibodies can be prepared by the techniques originally described inKohler 1975, Nature 256, 495, and Galfré 1981, Meth. Enzymol. 73, 3,which comprise the fusion of mouse myeloma cells to spleen cells derivedfrom immunized mammals. The antibodies can be used, for example, for theimmunoprecipitation, immunolocalization or purification (e.g., byaffinity chromatography) of the polypeptides of the invention as well asfor the monitoring of the presence of said variant polypeptides, forexample, in recombinant organisms, and for the identification ofproteins or compounds interacting with the proteins according to theinvention.

Moreover, the present invention contemplates a non-human transgenicorganism comprising the polynucleotide or the vector of the presentinvention.

Preferably, the non-human transgenic organism is a plant, plant part, orplant seed. Preferred plants to be used for introducing thepolynucleotide or the vector of the invention are plants which arecapable of synthesizing fatty acids, such as all dicotyledonous ormonocotyledonous plants, algae or mosses. It is to be understood thathost cells derived from a plant may also be used for producing a plantaccording to the present invention. Preferred plants are selected fromthe group of the plant families Adelotheciaceae, Anacardiaceae,Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae,Bromeliaceae, Caricaceae, Cannabaceae, Convolvulaceae, Chenopodiaceae,Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagnaceae,Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae,Juglandaceae, Lauraceae, Leguminosae, Linaceae, Prasinophyceae orvegetable plants or ornamentals such as Tagetes. Examples which may bementioned are the following plants selected from the group consistingof: Adelotheciaceae such as the genera Physcomitrella, such as the genusand species Physcomitrella patens, Anacardiaceae such as the generaPistacia, Mangifera, Anacardium, for example the genus and speciesPistacia vera [pistachio], Mangifer indica [mango] or Anacardiumoccidentale [cashew], Asteraceae, such as the genera Calendula,Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta,Tagetes, Valeriana, for example the genus and species Calendulaofficinalis [common marigold], Carthamus tinctorius [safflower],Centaurea cyanus [cornflower], Cichorium intybus [chicory], Cynarascolymus [artichoke], Helianthus annus [sunflower], Lactuca sativa,Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa,Lactuca scariola L. var. integrata, Lactuca scariola L. var.integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [african or french marigold], Apiaceae, such as the genusDaucus, for example the genus and species Daucus carota [carrot],Betulaceae, such as the genus Corylus, for example the genera andspecies Corylus avellana or Corylus colurna [hazelnut], Boraginaceae,such as the genus Borago, for example the genus and species Boragoofficinalis [borage], Brassicaceae, such as the genera Brassica,Melanosinapis, Sinapis, Arabadopsis, for example the genera and speciesBrassica napus, Brassica rapa ssp. [oilseed rape], Sinapis arvensisBrassica juncea, Brassica juncea var. juncea, Brassica juncea var.crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Melanosinapis communis [mustard], Brassica oleracea [fodderbeet] or Arabidopsis thaliana, Bromeliaceae, such as the genera Anana,Bromelia (pineapple), for example the genera and species Anana comosus,Ananas ananas or Bromelia comosa [pineapple], Caricaceae, such as thegenus Carica, such as the genus and species Carica papaya [pawpaw],Cannabaceae, such as the genus Cannabis, such as the genus and speciesCannabis sativa [hemp], Convolvulaceae, such as the genera Ipomea,Convolvulus, for example the genera and species Ipomoea batatus, Ipomoeapandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoeafastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus[sweet potato, batate], Chenopodiaceae, such as the genus Beta, such asthe genera and species Beta vulgaris, Beta vulgaris var. altissima, Betavulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Betavulgaris var. conditiva or Beta vulgaris var. esculenta [sugarbeet],Crypthecodiniaceae, such as the genus Crypthecodinium, for example thegenus and species Cryptecodinium cohnii, Cucurbitaceae, such as thegenus Cucurbita, for example the genera and species Cucurbita maxima,Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin/squash],Cymbellaceae such as the genera Amphora, Cymbella, Okedenia,Phaeodactylum, Reimeria, for example the genus and species Phaeodactylumtricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis,Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium,Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon,Skottsbergia, for example the genera and species Ceratodon antarcticus,Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpureus,Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon,purpureus spp. stenocarpus, Ceratodon purpureus var. rotundifolius,Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis,Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum,Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule,Ditrichum giganteum, Ditrichum heteromallum, Ditrichum lineare,Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichumpallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillumvar. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichumtortile, Distichium capillaceum, Distichium hagenii, Distichiuminclinatum, Distichium macounii, Eccremidium floridanum, Eccremidiumwhiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridiumalternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridiumravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodonborealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus,Elaeagnaceae such as the genus Elaeagnus, for example the genus andspecies Olea europaea [olive], Ericaceae such as the genus Kalmia, forexample the genera and species Kalmia latifolia, Kalmia angustifolia,Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida [mountain laurel], Euphorbiaceae suchas the genera Manihot, Janipha, Jatropha, Ricinus, for example thegenera and species Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcin, Manihot manihot, Manihotmelanobasis, Manihot esculenta [manihot] or Ricinus communis [castor-oilplant], Fabaceae such as the genera Pisum, Albizia, Cathormion,Feuillea, lnga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine,Dolichos, Phaseolus, Soja, for example the genera and species Pisumsativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albiziajulibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis,Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuilleaberteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acaciajulibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosajulibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck,Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck,Mimosa speciosa [silk tree], Medicago sativa, Medicago falcata, Medicagovaria [alfalfa], Glycine max Dolichos soja, Glycine gracilis, Glycinehispida, Phaseolus max, Soja hispida or Soja max [soybean], Funariaceaesuch as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella,Physcomitrium, for example the genera and species Aphanorrhegmaserratum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodonbonplandii, Entosthodon californicus, Entosthodon drummondii,Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus,Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon tucsoni,Funaria americana, Funaria bolanderi, Funaria calcarea, Funariacalifornica, Funaria calvescens, Funaria convoluta, Funaria flavicans,Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var.convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var.utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia,Funaria muhlenbergii, Funaria orcuttii, Funaria plano-convexa, Funariapolaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funariasonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrellacalifornica, Physcomitrella patens, Physcomitrella readeri,Physcomitrium australe, Physcomitrium californicum, Physcomitriumcollenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum,Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitriumflexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum,Physcomitrium immersum, Physcomitrium kellermanii, Physcomitriummegalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var.serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitriumsubsphaericum, Physcomitrium washingtoniense, Geraniaceae, such as thegenera Pelargonium, Cocos, Oleum, for example the genera and speciesCocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut],Gramineae, such as the genus Saccharum, for example the genus andspecies Saccharum officinarum, Juglandaceae, such as the genera Juglans,Wallia, for example the genera and species Juglans regia, Juglansailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea,Juglans bixbyi, Juglans californica, Juglans hindsii, Juglansintermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa,Juglans nigra or Wallia nigra [walnut], Lauraceae, such as the generaPersea, Laurus, for example the genera and species Laurus nobilis [bay],Persea americana, Persea gratissima or Persea persea [avocado],Leguminosae, such as the genus Arachis, for example the genus andspecies Arachis hypogaea [peanut], Linaceae, such as the genera Linum,Adenolinum, for example the genera and species Linum usitatissimum,Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense or Linum trigynum [linseed], Lythrarieae, suchas the genus Punica, for example the genus and species Punica granatum[pomegranate], Malvaceae, such as the genus Gossypium, for example thegenera and species Gossypium hirsutum, Gossypium arboreum, Gossypiumbarbadense, Gossypium herbaceum or Gossypium thurberi [cotton],Marchantiaceae, such as the genus Marchantia, for example the genera andspecies Marchantia berteroana, Marchantia foliacea, Marchantiamacropora, Musaceae, such as the genus Musa, for example the genera andspecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana],Onagraceae, such as the genera Camissonia, Oenothera, for example thegenera and species Oenothera biennis or Camissonia brevipes [eveningprimrose], Palmae, such as the genus Elacis, for example the genus andspecies Elaeis guineensis [oil palm], Papaveraceae, such as the genusPapaver, for example the genera and species Papaver orientale, Papaverrhoeas, Papaver dubium [poppy], Pedaliaceae, such as the genus Sesamum,for example the genus and species Sesamum indicum [sesame], Piperaceae,such as the genera Piper, Artanthe, Peperomia, Steffensia, for examplethe genera and species Piper aduncum, Piper amalago, Piperangustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum,Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata,Peperomia elongata, Piper elongatum, Steffensia elongata [cayennepepper], Poaceae, such as the genera Hordeum, Secale, Avena, Sorghum,Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum, for examplethe genera and species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras,Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeumsativum, Hordeum secalinum [barley], Secale cereale [rye], Avena sativa,Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida[oats], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghumvulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryzasativa, Oryza latifolia [rice], Zea mays [maize], Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, for example the genus and species Porphyridiumcruentum, Proteaceae, such as the genus Macadamia, for example the genusand species Macadamia intergrifolia [macadamia], Prasinophyceae such asthe genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus, for example the genera and speciesNephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata,Ostreococcus tauri, Rubiaceae such as the genus Cofea, for example thegenera and species Cofea spp., Coffea arabica, Coffea canephora orCoffea liberica [coffee], Scrophulariaceae such as the genus Verbascum,for example the genera and species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [mullein], Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon, for example the genera and speciesCapsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens[pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, forexample the genus and species Theobroma cacao [cacao] or Theaceae, suchas the genus Camellia, for example the genus and species Camelliasinensis [tea]. In particular preferred plants to be used as transgenicplants in accordance with the present invention are oil fruit cropswhich comprise large amounts of lipid compounds, such as peanut, oilseedrape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oilplant, olive, sesame, Calendula, Punica, evening primrose, mullein,thistle, wild roses, hazelnut, almond, macadamia, avocado, bay,pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm,coconut, walnut) or crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops. Preferred plants according to the invention are oilcrop plants such as peanut, oilseed rape, canola, sunflower, safflower,poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica,evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oilpalm, coconut). Especially preferred are sunflower, safflower, tobacco,mullein, sesame, cotton, pumpkin/squash, poppy, evening primrose,walnut, linseed, hemp, thistle or safflower. Very especially preferredplants are plants such as safflower, sunflower, poppy, evening primrose,walnut, linseed, or hemp.

Preferred mosses are Physcomitrella or Ceratodon. Preferred algae areIsochrysis, Mantoniella, Ostreococcus or Crypthecodinium, andalgae/diatoms such as Phaeodactylum or Thraustochytrium. Morepreferably, said algae or mosses are selected from the group consistingof: Emiliana, Shewanella, Physcomitrella, Thraustochytrium, Fusarium,Phytophthora, Ceratodon, Isochrysis, Aleurita, Muscarioides,Mortierella, Phaeodactylum, Cryphthecodinium, specifically from thegenera and species Thallasiosira pseudonona, Euglena gracilis,Physcomitrella patens, Phytophtora infestans, Fusarium graminaeum,Cryptocodinium cohnii, Ceratodon purpureus, Isochrysis galbana, Aleuritafarinosa, Thraustochytrium sp., Muscarioides viallii, Mortierellaalpina, Phaeodactylum tricornutum or Caenorhabditis elegans orespecially advantageously Phytophtora infestans, Thallasiosirapseudonona and Cryptocodinium cohnii.

Transgenic plants may be obtained by transformation techniques aselsewhere in this specification. Preferably, transgenic plants can beobtained by T-DNA-mediated transformation. Such vector systems are, as arule, characterized in that they contain at least the vir genes, whichare required for the Agrobacterium-mediated transformation, and thesequences which delimit the T-DNA (T-DNA border). Suitable vectors aredescribed elsewhere in the specification in detail.

Also encompassed are transgenic non-human animals comprising the vectoror polynucleotide of the present invention. Preferred non-humantransgenic animals envisaged by the present invention are fish, such asherring, salmon, sardine, redfish, eel, carp, trout, halibut, mackerel,zander or tuna.

However, it will be understood that dependent on the non-humantransgenic organism specified above, further, enzymatic activities maybe conferred to the said organism, e.g., by recombinant technologies.Accordingly, the present invention, preferably, envisages a non-humantransgenic organism specified above which in addition to thepolynucleotide of the present invention comprises polynucleotidesencoding such desaturases and/or elongases as required depending on theselected host cell. Preferred desaturases and/or elongases which shallbe present in the organism are at least one enzyme selected from thegroup of desaturases and/or elongases or the combinations specificallyrecited elsewhere in this specification (see above and Tables 3, 4 and5).

Furthermore, the present invention encompasses a method for themanufacture of polyunsaturated fatty acids comprising:

-   a) cultivating the host cell of the invention under conditions which    allow for the production of polyunsaturated fatty acids in said host    cell;-   b) obtaining said polyunsaturated fatty acids from the said host    cell.

The term “polyunsaturated fatty acids (PUFA)” as used herein refers tofatty acids comprising at least two, preferably, three, four, five orsix, double bonds. Moreover, it is to be understood that such fattyacids comprise, preferably from 18 to 24 carbon atoms in the fatty acidchain. More preferably, the term relates to long chain PUFA (LCPUFA)having from 20 to 24 carbon atoms in the fatty acid chain. Preferredunsaturated fatty acids in the sense of the present invention areselected from the group consisting of DGLA 20:3 (8,11,14), ARA 20:4(5,8,11,14), iARA 20:4(8,11,14,17), EPA 20:5 (5,8,11,14,17), DPA 22:5(4,7,10,13,16), DHA 22:6 (4,7,10,13,16,19), 20:4 (8,11,14,17), morepreferably, arachidonic acid (ARA) 20:4 (5,8,11,14), eicosapentaenoicacid (EPA) 20:5 (5,8,11,14,17), and docosahexaenoic acid (DHA) 22:6(4,7,10,13,16,19). Thus, it will be understood that most preferably, themethods provided by the present invention pertaining to the manufactureof ARA, EPA or DHA. Moreover, also encompassed are the intermediates ofLCPUFA which occur during synthesis. Such intermediates are, preferably,formed from substrates by the desaturase or elongase activity of thepolypeptides of the present invention. Preferably, substrates encompassLA 18:2 (9,12), ALA 18:3(9,12,15), Eicosadienoic acid 20:2 (11,14),Eicosatrienoic acid 20:3 (11,14,17)), DGLA 20:3 (8,11,14), ARA 20:4(5,8,11,14), eicosatetraenoic acid 20:4 (8,11,14,17), Eicosapentaenoicacid 20:5 (5,8,11,14,17), Docosahexapentanoic acid 22:5 (7,10,13,16,19).

The term “cultivating” as used herein refers maintaining and growing thehost cells under culture conditions which allow the cells to produce thesaid polyunsaturated fatty acid, i.e. the PUFA and/or LCPUFA referred toabove. This implies that the polynucleotide of the present invention isexpressed in the host cell so that the desaturase and/or elongaseactivity is present. Suitable culture conditions for cultivating thehost cell are described in more detail below.

The term “obtaining” as used herein encompasses the provision of thecell culture including the host cells and the culture medium as well asthe provision of purified or partially purified preparations thereofcomprising the polyunsaturated fatty acids, preferably, ARA, EPA, DHA,in free or in -CoA bound form, as membrane phospholipids or astriacylglyceride estres. More preferably, the PUFA and LCPUFA are to beobtained as triglyceride esters, e.g., in form of an oil. More detailson purification techniques can be found elsewhere herein below.

The host cells to be used in the method of the invention are grown orcultured in the manner with which the skilled worker is familiar,depending on the host organism. Usually, host cells are grown in aliquid medium comprising a carbon source, usually in the form of sugars,a nitrogen source, usually in the form of organic nitrogen sources suchas yeast extract or salts such as ammonium sulfate, trace elements suchas salts of iron, manganese and magnesium and, if appropriate, vitamins,at temperatures of between 0° C. and 100° C., preferably between 10° C.and 60° C. under oxygen or anaerobic atmosphere depedent on the type oforganism. The pH of the liquid medium can either be kept constant, thatis to say regulated during the culturing period, or not. The culturescan be grown batchwise, semibatchwise or continuously. Nutrients can beprovided at the beginning of the fermentation or administerdsemicontinuously or continuously: The produced PUFA or LCPUFA can beisolated from the host cells as described above by processes known tothe skilled worker, e.g., by extraction, distillation, crystallization,if appropriate precipitation with salt, and/or chromatography. It mightbe required to disrupt the host cells prior to purification. To thisend, the host cells can be disrupted beforehand. The culture medium tobe used must suitably meet the requirements of the host cells inquestion. Descriptions of culture media for various microorganisms whichcan be used as host cells according to the present invention can befound in the textbook “Manual of Methods for General Bacteriology” ofthe American Society for Bacteriology (Washington D.C., USA, 1981).Culture media can also be obtained from various commercial suppliers.All media components are sterilized, either by heat or by filtersterilization. All media components may be present at the start of thecultivation or added continuously or batchwise, as desired. If thepolynucleotide or vector of the invention which has been introduced inthe host cell further comprises an expressible selection marker, such asan antibiotic resistance gene, it might be necessary to add a selectionagent to the culture, such as a antibiotic in order to maintain thestability of the introduced polynucleotide. The culture is continueduntil formation of the desired product is at a maximum. This is normallyachieved within 10 to 160 hours. The fermentation broths can be useddirectly or can be processed further. The biomass may, according torequirement, be removed completely or partially from the fermentationbroth by separation methods such as, for example, centrifugation,filtration, decanting or a combination of these methods or be leftcompletely in said broth. The fatty acid preparations obtained by themethod of the invention, e.g., oils, comprising the desired PUFA orLCPUFA as triglyceride esters are also suitable as starting material forthe chemical synthesis of further products of interest. For example,they can be used in combination with one another or alone for thepreparation of pharmaceutical or cosmetic compositions, foodstuffs, oranimal feeds. Chemically pure triglycerides comprising the desired PUFAor LCPUFA can also be manufactured by the methods described above. Tothis end, the fatty acid preparations are further purified byextraction, distillation, crystallization, chromatography orcombinations of these methods. In order to release the fatty acidmoieties from the triglycerides, hydrolysis may be also required. Thesaid chemically pure triglycerides or free fatty acids are, inparticular, suitable for applications in the food industry or forcosmetic and pharmacological compositions.

Moreover, the present invention relates to a method for the manufactureof poly-unsaturated fatty acids comprising:

-   a) cultivating the non-human transgenic organism of the invention    under conditions which allow for the production of poly-unsaturated    fatty acids in said non-human transgenic organism; and-   b) obtaining said poly-unsaturated fatty acids from the said    non-human transgenic organism.

Further, it follows from the above that a method for the manufacture ofan oil, lipid or fatty acid composition is also envisaged by the presentinvention comprising the steps of any one of the aforementioned methodsand the further step of formulating PUFA or LCPUFA as oil, lipid orfatty acid composition. Preferably, said oil, lipid or fatty acidcomposition is to be used for feed, foodstuffs, cosmetics ormedicaments. Accordingly, the formulation of the PUFA or LCPUFA shall becarried out according to the GMP standards for the individual envisagedproducts. For example, an oil may be obtained from plant seeds by an oilmill. However, for product safety reasons, sterilization may be requiredunder the applicable GMP standard. Similar standards will apply forlipid or fatty acid compositions to be applied in cosmetic orpharmaceutical compositions. All these measures for formulating oil,lipid or fatty acid compositions as products are comprised by theaforementioned manufacture.

The term “oil” refers to a fatty acid mixture comprising unsaturatedand/or saturated fatty acids which are esterified to triglycerides.Preferably, the triglycerides in the oil of the invention comprise PUFAor LCPUFA as referred to above. The amount of esterified PUFA and/orLCPUFA is, preferably, approximately 30%, a content of 50% is morepreferred, a content of 60%, 70%, 80% or more is even more preferred.The oil may further comprise free fatty acids, preferably, the PUFA andLCPUFA referred to above. For the analysis, the fatty acid content canbe, e.g., determined by GC analysis after converting the fatty acidsinto the methyl esters by transesterification. The content of thevarious fatty acids in the oil or fat can vary, in particular dependingon the source. The oil, however, shall have a non-naturally occurringcomposition with respect to the PUFA and/or LCPUFA composition andcontent. It will be understood that such a unique oil composition andthe unique esterification pattern of PUFA and LCPUFA in thetriglycerides of the oil shall only be obtainable by applying themethods of the present invention specified above. Moreover, the oil ofthe invention may comprise other molecular species as well.Specifically, it may comprise minor impurities of the polynucleotide orvector of the invention. Such impurities, however, can be detected onlyby highly sensitive techniques such as PCR.

The contents of all references cited throughout this application areherewith incorporated by reference in general and with respect to theirspecific disclosure content referred to above.

FIGURES

FIG. 1 shows a schematical figure of the different enzymatic activitiesleading to the production of ARA, EPA and DHA.

FIG. 2 shows a yeast expression experiment with feeding of 22:5n-3 inthe prescence (A) and absence (B) of d4Des(Eh).

FIG. 3 shows a yeast expression experiment with feeding of 20:3n-3 inthe prescence (A) and absence (B) of d8Des(Eh).

FIG. 4 shows a yeast expression experiment with feeding of 18:3n-3 inthe prescence (A) and absence (B) of d9Elo(Eh).

FIG. 5 shows a yeast expression experiment with feeding of 18:3n-6 (GLA)and 18:4n-3 (SDA) in the prescence (A) and absence (B) of d5Elo(Eh).

FIG. 6 shows a yeast expression experiment with feeding of 20:4n-6 (ARA)and 20:5n-3 (EPA) in the prescence (A) and absence (B) of d5Elo(Eh).

FIG. 7 shows the expression of d9Elo(Eh) in seeds of two Arabidopsisevents. As control seeds not expression d9Elo(Eh) are shown (WT).

FIG. 8 shows the Acyl-CoA analysis of mature Arabidopsis seeds from bothevents expressing the d9Elo(Eh) in comparison to seeds not expressingd9Elo(Eh) (Col0)).

FIG. 9 shows the expression of d9Elo(Eh), d8Des(Eh) and d5Des(Eh) inseeds of various Arabidopsis events.

FIG. 10 shows gas chromatographic analysis of mature Arabidopsis seedstransformed with the construct OstELO5EmD4. Peaks were quantified andlisted in the table below. The products of d5Elo(Ot) and d4Des(Eh)activity are 22:6n-3 (DHA).

FIG. 11 is a comparison between two d4-desaturases (Tc and Eh) showingthat d4Des(Eh) is different from known d4-desaturases in producing ahigh ratio of DHA:DPA.

FIG. 12 shows the expression of d5Elo(Eh) in seeds of variousArabidopsis events.

FIG. 13 is a comparison between three different d6-desaturases and thesubstrate specificity of d5Des(Eh).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example 1: Organism and Culture Conditions

Emiliana huxleyi was grown as described in Sciandra et al. (2003) MarineEcology Progress Series 261:111-122 with following conditions:

Growth in 50 ml inconical flasks using K/2 medium (Keller et al. (1987)Journal of Phycology 23:633-638). The flasks were placed in a growthchamber at a temperature of 17±0.1° C. under 14L:10D irradiance. Lightwas provided by fluorescent lamps giving a photon fluxdensity (400 to700 nm) of 170 μmol photon m-2 s-1.

Example 2: Cloning of Novel Desaturase and Elongase Sequences

RNA from cells grown as described under Example 1 was extracted usingthe RNA-extraction Kit from Qiagen, a RACE-library was generated usingthe RACE-Kit from Clontech. From the RACE-library sequences fordesaturase and elongases were amplified with PCR using following primerpairs and PCR conditions.

PCR reaction (50 μl):

5.00 μl Template cDNA

5.00 μl 10× Puffer (Advantage-Polymerase)+25 mM MgCl₂

5.00 μl 2 mM dNTP

1.25 μl je Primer (10 pmol/μL)

0.50 μl Advantage-Polymerase

The Advantage polymerase mix from Clontech was used.

Reaction conditions of the PCR:

Annealing: 1 min 55° C.

Denaturation: 1 min 94° C.

Elongation: 2 min 72° C.

Cycles: 35

Primer pairs used in PCR:

Name Primer pair (5′ orientation) SEQ ID NO. Eh4ffCCATGGGAGGCGCCGGCGCGAG 11 Eh4rv CTAGTCCGCCTTGAGGTTCTC 12 Eh5ffACCATGTGCAAGGCGAGCGGCCT 13 Eh5rv TCACCAATCATGAGGAAGGT 14 Eh8ffCCATGGGCAAGGGCGGCAACGC 15 Eh8rv GGGCAGAGATGCCGCACTAG 16 Eh9ffACCATGCTCGATCGCGCCTCGTC 17 Eh9rv TCACAGCGCCTTGCGGGTAGC 18

The PCR reactions resulted in following polynucleotide sequences:

Gene Activity Length in bp SEQ ID NO. D4Des(Eh) D4-desaturase 1280 5D8Des(Eh) D8-desaturase 1256 1 D9Elo(Eh) D9-elongase  804 3 D5Elo(Eh)Multi-elongase  921 7

A list of identified full-length coding sequences is shown in Table 1.

TABLE 1 List of full-length coding sequences and deduced amino acidsequences SEQ ID NO: Gene Coding sequence in bp Amino acid sequence 1D8Des(Eh) 1254 417 3 D9Elo(Eh) 801 266 5 D4Des(Eh) 1278 425 7 D5Elo(Eh)918 305

Open reading frames as shown in Table 1 were cloned into the pESC(Leu)vector from Stratagene according to manufactures reaction conditions.Reactions were transformed into E. coli DH5a and plasmid DNA wasisolated. The plasmids pESC-d4Des(Eh), pESC-d8Des(Eh), pESC-d9Elo(Eh),pESC-d5Elo(Eh) were then used for yeast transformation.

Example 3: Yeast Transformation and Growth Conditions

S. cerevisiae strain INVSC from Invitrogen was transformed with theconstructs pESC-d4Des(Eh), pESC-d8Des(Eh), pESC-d9Elo(Eh),pESC-d5Elo(Eh) and pESC using the S. C. EasyComp Transformation Kit(Invitrogen, Carlsbad, Calif.) with selection on leucine-deficientmedium.

Yeast were grown after transformation in complete medium containing allamino acids and nucleotides. Then yeast were plated on different mediumcontaining either the complete medium (SD) or the complete mediumlacking leucine (SD-Leu). Only yeast containing pESC-d4Des(Eh),pESC-d8Des(Eh), pESC-d9Elo(Eh), pESC-d5Elo(Eh) or pESC vector can growon this medium.

Example 4: Functional Expression of Desaturases and Elongases in Yeastand Gas Chromatographic Analysis

Yeast cells containing the respective pESC plasmids as prepared abovewere incubated 12 h in liquid DOB-U medium at 28° C., 200 rpm inkubiertand than additional 12 h in induction medium (DOB-U+2% (w/v)galactose+2% (w/v) raffinose). To the induction medium 250 μM of therespecitve fatty acids were added to check for enzyme activity andspecificity.

Yeast cells were analyzed as following:

Yeast cells from induction medium were harvested by centrifugation(100×g, 5 min, 20° C.) and washed with 100 mM NaHCO₃, pH 8,0, to removeresidual fatty acids. From the yeast pellet a total extract of fattyacid methylesters (FAME) was generated by adding 2 ml 1 N methanolicsulfuric acid and 2% (v/v) Dimethoxypropan for 1 h at 80° C. FAME wereextracted two times with Petrolether (PE). Not derivased fatty acidswere removed by washing with 2 ml 100 mM NaHCO₃, pH 8.0 and 2 ml Aquadent. The PE-phases were dried with Na₂SO₄ and eluted in 100 μl PE. Thesamples were then separated with a DB-23-column (30 m, 0.25 mm, 0.25 μm,Agilent) in a Hewlett-Packard 6850-machine with FID using followingconditions: oven temperature 50° C. to 250° C. with a rate of 5° C./minand finally 10 min at 250° C.

The identification of the fatty acids was done using the retention timesof known fatty acid standards (Sigma). The method is described e.g. inNapier and Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al.,2001, Journal of Experimental Botany. 52(360):1581-1585, Sperling etal., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al.,1998, FEBS Letters. 439(3):215-218.

Example 5: Functional Characterization of d4Des(Eh)

As described above d4Des(Eh) was functionally characterized in yeast.The result of the analysis is shown in FIG. 2. Yeast transformed withpESC-d4Des(Eh) was compared to yeast transformed with pESC (control)while feeding the yeast cells with the fatty acid DPA 22:5n-3. Based onthis comparison pESC-d4Des(Eh) exhibits d4-desaturase activity as in thecontrol no 22:6 is observed. Therefore d4Des(Eh) is a functionald4-desaturase.

Example 6: Functional Characterization of d8Des(Eh)

As described above d8Des(Eh) was functionally characterized in yeast.The result of the analysis is shown in FIG. 3. Yeast transformed withpESC-d8Des(Eh) was compared to yeast transformed with pESC (control)while feeding the fatty acid 20:3n-3. Based on this comparison a newfatty acid was formed compared to the control, which is 20:4n-3. Theformation of this fatty acid proves that d8Des(Eh) was functionallyexpressed and has d8-desaturase activity. The conversion rate of 20:3n-3to 20:4n-3 was 5%.

Example 7: Functional Characterization of d9Elo(Eh)

As described above d9Elo(Eh) was functionally characterized in yeast.The result of the analysis is shown in FIG. 4. Yeast transformed withpESC-d9Elo(Eh) was compared to yeast transformed with pESC (control)while feeding the fatty acids 18:3n-3 (ALA) or 18:2 (LA). Based on thiscomparison a new fatty acid was formed compared to the control, which is20:3n-3 or 20:2n-6, respectively. The formation of these fatty acidsproves that d9Elo(Eh) was functionally expressed and has d9-elongaseactivity. The conversion rate of 18:3n-3 to 20:3n-3 was 17%, theconversion rate of 18:2n-6 to 20:2n-6 was 49%.

Example 8: Functional Characterization of d5Elo(Eh)

As described above d5Elo(Eh) was functionally characterized in yeast.The result of the analysis is shown in FIGS. 5 and 6. Yeast transformedwith pESC-d5Elo(Eh) was compared to yeast transformed with pESC(control) while feeding the fatty acids 18:3n-6 (GLA), 18:4 (SDA) or20:4n-6 (ARA), 20:5n-3 (EPA), respectively. Based on this comparison newfatty acids formation was observed when compared to the control, whichis 20:3n-6 or 20:4n-3 when fed GLA or SDA and 22:4n-6 or 22:5n-3 whenfed ARA or EPA, respectively. The formation of these fatty acids provesthat d5Elo(Eh) was functionally expressed and has d5-elongase activity.The conversion rate of GLA was 13%, the conversion rate of 18:4n-3 was30%, the conversion rate of ARA was 38% and the conversion rate of EPAwas 30%. Surprisingly the elongase used a wide variety of substrates ofelongation. The specification indicates a multifunctional elongaseactivity with higher specificities for omega3 fatty acids.

Example 9: Expression of Novel Elongases from Emiliana huxleyi in Plants

The novel desaturases and elongases were cloned into a planttransformation vector as described in WO2003/093482, WO2005/083093 orWO2007/093776. Exemplary suitable combinations of genes are described inTable 2, 3 and 4.

TABLE 2 Gene combinations for the production of ARA. Gene Activity SEQID NO: D6Des(Ot) Δ6-Desaturase 19 D6Elo(Pp) Δ6-Elongase 21 D5Des(Eh)Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25D8Des(Eh) Δ8-Desaturase 1 D9Elo(Eh) Δ9-Elongase 3

TABLE 3 Gene combinations for the production of EPA. Gene Activity SEQID NO: D6Des(Ot) Δ6-Desaturase 19 D5Elo(Eh) Δ5-Elongase 7 D5Des(Eh)Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25o3-Des(Pi) Omega 3-Desaturase 27 D15Des(Cp) Δ15-Desaturase 29 D8Des(Eh)Δ8-Desaturase 1 D9Elo(Eh) Δ9-Elongase 3

TABLE 4 Gene combinations for the production of DHA. Gene Activity SEQID NO: D6Des(Ot) Δ6-Desaturase 19 D5Elo(Eh) Δ5-Elongase 7 D5Des(Eh)Δ5-Desaturase 9 D12Des(Ps) Δ12-Desaturase 23 D6Elo(Tp) Δ6-Elongase 25ω3-Des(Pi) Omega 3-Desaturase 27 D15Des(Cp) Δ15-Desaturase 29 D5Elo(Ot)Δ5-elongase 31 D4Des(Eh) Δ4-desaturase 5 D8Des(Eh) Δ8-Desaturase 1D9Elo(Eh) Δ9-Elongase 3

Based on the gene combinations as described in Table 2, Table 3 or Table4 following combinations were designed:

-   -   AP2:        LuCnl-d5Des(Eh)_LuCnl-d8Des8Eh)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d9Elo(Eh)    -   OstELO5EmD4:        VfUSP-d6Elo(Pp)_LuCnl-d5Des8Tc)_VfSBP-d6Des(Ot)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d5Elo(Ot)_LuCnl-d4Des(Eh)    -   OstELO5TcD4:        VfUSP-d6Elo(Pp)_LuCnl-d5Des8Tc)_VfSBP-d6Des(Ot)_Napin-o3Des(Pi)_Napin-d12Des(Ps)_LuCnl-d5Elo(Ot)_LuCnl-d4Des(Tc)

Transgenic rapeseed lines were generated as described in Deblaere et al,1984, Nucl. Acids. Res. 13, 4777-4788 and seeds of transgenic rapeseedplants are analyzed as described in Qiu et al. 2001, J. Biol. Chem. 276,31561-31566.

Transgenic Arabidopsis plants were generated as described in Bechtholdtet al. 1993 C. R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199. Seedsof transgenic Arabidopsis plants expressing d9Elo(Eh) by using theseed-specific promoter Glycinin from soybean (Lelievre et al. (1992)Plant Physiol 98:387-391) were analyzed by gas chromatography (FIG. 7).Compared to non-transgenic control plants (WT) there are changes in thefatty acid profile, proving that d9Elo(Eh) was functionally expressionin seeds. The major shifts in the fatty acid profile is directed to a 10fold increase in the fatty acid 20:2n-6 and 20:3n-3 (FIG. 7). Therefored9Elo(Eh) exhibits a Δ9-elongase activity, which is consistent with theyeast characterization. Further, the levels of 18:2 and ALA in thetransgenic events expressing d9Elo(Eh) are lowered compared to WT, asthese fatty acids are direct substrates for the d9Elo(Eh). Further, theendogenous elongation system in the plant is unchanged as levels of 20:1and 22:1 are similar between transgenic plants expression d9Elo(Eh) andWT control. This indicates that the expression of d9Elo(Eh) does notdisturb endogenous elongation process, but delivers additional activity.

To further prove the activity of d9Elo(Eh) expressed in seeds ofArabidopsis thaliana AcylCoA-measurements were done. Substrates andproducts of the d9Elo(Eh) elongation reaction are AcylCoA-esters, whichare then further incorporated into triacylglycerides (oil). The analysisof the acylCoA-pool reveals the formation and flux of the elongationreaction.

FIG. 8 summarizes the AcylCoA measurements for Arabidopsis eventexpressing d9Elo(Eh) in comparison to controls not expressing d9Elo(Eh)(Col0). The change in the chromatogram is indicated by a star. At thisposition a massive amount of 20:2n-6 is detected, which is much lower inthe control. The conditions for separation of the fatty acid CoA-estersdoes not allow the detection of 20:3n-3 as this CoA ester is notseparated from 18:3CoA.

The massive occurrence of 20:2n-6-CoA proves the expression of d9Elo(Eh)as this is the direct product of its enzymatic activity.

Further, transgenic Arabidopsis lines have been generated to validatethe activity of d8Des(Eh) and d5Des(Eh). Vector AP2 has been constructedaccording to standard molecular biology steps as described inWO2003/093482, WO2005/083093, WO2007/093776 or WO2009/016202 andtransformed into Arabidopsis thaliana as described above. Analysis oftransgenic seeds is shown in FIG. 9. The products of d9Elo(Eh) are 20:2and 20:3n-3.

Further, transgenic Arabidopsis lines have been generated to validatethe activity of d4Des(Eh). Construct OstELO5EmD4 was transformed intoArabidopsis as described above and seeds of a number of individual lineshave been analyzed by gas chromatography (FIG. 10). The activity ofd4Des(Eh) is demonstrated by the formation of DHA 22:6 (last column).All lines show the production of DHA with levels of up to 4.7%. Ofspecial interest is the ratio of DHA to DPA. Surprisingly the ratio ofd4Des(Eh) is much higher than in d4-desaturases known in the art. Acomparison against the d4-desaturase from Thraustochytrium ssp. ofWO2002/026946 is shown in FIG. 11. The enzyme from Thraustochytrium ssp.showed so far highest levels of DHA (WO2005/083093), but with anunfavorable ratio of DPA to DHA. A high ratio of DHA:DPA is for thecommercial use of such oils of importance.

Further, transgenic Arabidopsis lines have been generated to validatethe activity of d5Elo(Eh). Construct EmELO5TcD4 was transformed intoArabidopsis as described above and seeds of a number of individual lineshave been analyzed by gas chromatography (FIG. 12). The activity ofd5Elo(Eh) is demonstrated by the formation of DPA 22:5 and DHA 22:6.Most lines show the production of these two fatty acids, proofing thatd5Elo(Em) is functionally expressed in the seeds.

Further, transgenic Arabidopsis lines have been generated to validatethe activity and substrate specificity of d5Des(Eh). For this purposetwo Δ6-desaturases were selected based on their different substratespecificity. The borageΔ6 is expected to use phosphatidylcholin-18:2 assubstrate (WO96/21022), whereas the Ostreococcus Δ6 (OstrΔ6) usesAcyl-CoA ester (WO2005/012316). In combination with the d6-elongase fromPhyscomitrella patens (WO2001/059128) both d6-desaturases produce DGLAor 20:4n-3, respectively. The ratio of ARA to EPA is for the borageΔ62.9, for the OstrΔ6 2.3. It is noted that the use of OstrΔ6 results in3-4 times higher levels of products compared to the borageΔ6. Thefurther combination of the d5Des(Eh) resulted in the production of ARAand EPA, demonstrating the functionality of the d5Des(Eh). Theconversion of d5Des(Eh) of DGLA to ARA is 29% (borageΔ6) or 47%(OstrΔ6). For 20:4n-3 to EPA it is 33% (borageΔ6) or 26% (OstrΔ6).

Based on these results it is concluded that for Acyl-CoA substratesd5Des(Eh) is specific for the omega6 fatty acid DGLA. This is a novelsubstrate specificity not observed in the state of the artd5-desaturases.

REFERENCE LIST

-   Arondel, V., Lemieux, B., Hwang, I., Gibson, S., Goodman, H. M., and    Somerville, C. R. (1992). Map-based cloning of a gene controlling    omega-3 fatty acid desaturation in Arabidopsis. Science 258,    1353-1355.-   Broadwater, J. A., Whittle, E., and Shanklin, J. (2002).    Desaturation and hydroxylation. Residues 148 and 324 of Arabidopsis    FAD2, in addition to substrate chain length, exert a major influence    in partitioning of catalytic specificity. J. Biol. Chem. 277,    15613-15620.-   Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998b).    Catalytic plasticity of fatty acid modification enzymes underlying    chemical diversity of plant lipids. Science 282, 1315-1317.-   Calvo, A. M., Gardner, H. W., and Keller, N. P. (2001). Genetic    connection between fatty acid metabolism and sporulation in    Aspergillus nidulans. J. Biol. Chem. 276, 25766-25774.-   Knutzon, D. S., Thurmond, J. M., Huang, Y. S., Chaudhary, S.,    Bobik, E. G., Jr., Chan, G. M., Kirchner, S. J., and Mukerji, P.    (1998). Identification of Delta5-dehydratase from Mortierella alpina    by heterologous expression in Bakers' yeast and canola. J. Biol.    Chem. 273, 29360-29366.-   Mantle, P. G. and Nisbet, L. J. (1976). Differentiation of Claviceps    purpurea in axenic culture. J. Gen. Microbiol. 93, 321-334.-   Mey, G., Oeser, B., Lebrun, M. H., and Tudzynski, P. (2002). The    biotrophic, non-appressorium-forming grass pathogen Claviceps    purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein    kinase for colonization of rye ovarian tissue. Mol. Plant Microbe    Interact. 15, 303-312.-   Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E., and    Browse, J. (1994). Arabidopsis FAD2 gene encodes the enzyme that is    essential for polyunsaturated lipid synthesis. Plant Cell 6,    147-158.-   Qi, B., Fraser, T., Mugford, S., Dobson, G., Sayanova, O., Butler,    J., Napier, J. A., Stobart, A. K., and Lazarus, C. M. (2004).    Production of very long chain polyunsaturated omega-3 and omega-6    fatty acids in plants. Nat. Biotechnol. 22, 739-745.-   Qiu, X., Hong, H., and McKenzie, SL. (2001) Identification of a    Delta 4 fatty acid desaturase from Thraustochytrium sp. involved in    the biosynthesis of docosahexanoic acid by heterologous expression    in Saccharomyces cerevisiae and Brassica juncea. J Biol Chem 276,    31561-6.-   Shanklin, J. and Cahoon, E. B. (1998). DESATURATION AND RELATED    MODIFICATIONS OF FATTY ACIDS1. Annu. Rev. Plant Physiol Plant Mol.    Biol. 49, 611-641.-   Tudzynski, P., Correia, T., and Keller, U. (2001). Biotechnology and    genetics of ergot alkaloids. Appl. Microbiol. Biotechnol. 57,    593-605.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

1. A polynucleotide comprising a nucleic acid sequence elected from thegroup consisting of: a) a nucleic acid sequence having a nucleotidesequence as shown in SEQ ID NOs: 1, 3, 5, 7 or 9; b) a nucleic acidsequence encoding a polypeptide having an amino acid sequence as shownin SEQ ID NOs: 2, 4, 6, 8 or 10; c) a nucleic acid sequence being atleast 70% identical to the nucleic acid sequence of a) or b), whereinsaid nucleic acid sequence encodes a polypeptide having desaturase orelongase activity; d) a nucleic acid sequence encoding a polypeptidehaving desaturase or elongase activity and having an amino acid sequencewhich is at least 82% identical to the amino acid sequence of any one ofa) to c); and e) a nucleic acid sequence which is capable of hybridizingunder stringent conditions to any one of a) to d), wherein said nucleicacid sequence encodes a polypeptide having desaturase or elongaseactivity.
 2. The polynucleotide of claim 1, wherein said polynucleotidefurther comprises an expression control sequence operatively linked tothe said nucleic acid sequence.
 3. The polynucleotide of claim 1,wherein said polynucleotide further comprises a terminator sequenceoperatively linked to the nucleic acid sequence.
 4. A vector comprisingthe polynucleotide of claim
 1. 5. A host cell comprising thepolynucleotide of claim
 1. 6. A method for the manufacture of apolypeptide comprising a) cultivating the host cell of claim 5 underconditions which allow for the production of the said polypeptide; andb) obtaining the polypeptide from the host cell of step a).
 7. Apolypeptide encoded by the polynucleotide of claim
 1. 8. A non-humantransgenic organism comprising the polynucleotide of claim
 1. 9. Thenon-human transgenic organism of claim 8, which is a plant, plant part,or plant seed.
 10. A method for the manufacture of polyunsaturated fattyacids comprising: a) cultivating the host cell of claim 5 underconditions which allow for the production of polyunsaturated fatty acidsin said host cell; and b) obtaining said polyunsaturated fatty acidsfrom the said host cell.
 11. A method for the manufacture ofpolyunsaturated fatty acids comprising: a) cultivating the non-humantransgenic organism of claim 8 under conditions which allow for theproduction of polyunsaturated fatty acids in said non-human transgenicorganism; and b) obtaining said polyunsaturated fatty acids from thesaid non-human transgenic organism.
 12. The method of claim 10, whereinsaid poly-unsaturated fatty acid is arachidonic acid (ARA),eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA).
 13. Themethod of claim 10, further comprising formulating the polyunsaturatedfatty acid as oil, lipid or fatty acid composition.
 14. The method ofclaim 13, wherein said oil, lipid or fatty acid composition is feed, afoodstuff, a cosmetic or a medicament.
 15. An Antibody or a fragmentthereof which specifically recognizes the polypeptide of claim 7.